HBV surface antigen (HBsAg) variants may impair diagnosis or allow the virus to escape vaccine-induced immunity and their circulation in the population can represent a Public Health threat. Their prevalence, however, is not yet completely established. Evidence indicates that amino acid substitutions within HBsAg can lead to conformational changes which allow mutated HBV to escape the vaccine-induced antibodies used in the screening tests. In such scenario, the aim of this study was to investigate the prevalence of HBV S-Gene escape mutants by sequencing the gene in a cohort of Ligurian patients monitored for viral load, genotype and drug resistance and to evaluate the risk of false negative HBsAg detection by routine screening tests. From 2007 to 2011, in 256 consecutive samples from Ligurian HBV positive patients sequencing assay for detection of RT/S-Gene mutations using Trugene HBV Genotyping kit (Siemens Healthcare Diagnostics Inc., Tarrytown, NY) was performed. Serological HBV tests and viral load were also performed. Analyzed sequences revealed G145R mutation in 8/256 (3.1%) examined sequences, it was alone in 5 patients and accompanied by other HBsAg mutations in 3 samples. HBsAg resulted undetectable by 3 of the 8 samples, derived from patients with multiple mutations: T126I-T131A-C139Y-E/D144G, T126I-M133L, and P120Q-T126I. The emergence of these mutants, at least the G145R, has already been addressed as a public health concern because of its capability of escaping the immune system. In the present study we point out a second aspect connected with their existence and with similar potential negative impact on public health, that is their capability of escape punctual detection.